Abstract
Using a functional genomics approach, four candidate genes (PtGT34A, PtGT34B, PtGT34C and PtGT34D) were identified in Pinus taeda that encode CAZy family GT34 glycosyltransferases involved in the synthesis of cell-wall xyloglucans and heteromannans. The full-length coding sequences of three orthologs PrGT34A, B, and C, were isolated from a xylem-specific cDNA library from the closely related Pinus radiata. PrGT34B is the ortholog of XXT1 and XXT2, the two main xyloglucan (1→6)-α-xylosyltransferases in Arabidopsis thaliana. PrGT34C is the ortholog of XXT5 in A. thaliana, which is also considered to be involved in the xylosylation of xyloglucans. PrGT34A is an ortholog of a fenugreek galactosyltransferase involved in galactomannan synthesis. Truncated coding sequences of the genes were cloned into plasmid vectors and expressed in a Sf9 insect cell-culture system. The heterologous proteins were purified and in vitro assays showed that when incubated with UDP-xylose and cellotetraose, cellopentaose or cellohexaose, PrGT34B showed xylosyltransferase activity, and when incubated with UDP-galactose and the same cellooligosaccharides, showed some galactosyltransferase activity. The ratio of xylosyl- to galactosyltransferase activity was 434:1. Hydrolysis of the galactosyltransferase reaction products with galactosidases showed the linkages formed were α-. Analysis of the products of PrGT34B by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry showed up to three xylosyl residues were transferred from UDP-xylose to cellohexaose. Heterologous proteins PrGT34A and PrGT34C showed no detectable enzymatic activity.This article is protected by copyright. All rights reserved.
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